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Updated: Sep 8, 2025
Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level
Published on: March 14, 2025
JNK knockdown enhances CAR-T cell cytotoxicity
Background:
Boosting the performance of chimeric antigen receptor T (CAR-T) cell therapy in solid tumors may provide a substantial advantage for cancer patients. Recognizing the vital role of the nuclear factor of activated T cells (NFAT) in T cell function, we hypothesized that the strategic regulation of NFAT activity by targeting c-Jun N-terminal Kinases (JNK) can bolster the tumor-eradicating potential of CAR-T cells.
Methods:
We developed a lentivirally encoded short-hairpin RNA (shRNA) for stable knockdown of JNK in CAR-T cells. CAR-T cells targeting human epidermal growth factor receptor 2 (HER2) were produced from human peripheral blood. Functionality was tested in vitro and in two xenograft models of human ovarian cancer.
Results:
JNK knockdown in CAR-T cells suppressed antigen-induced stimulation and helper T cell cytokine production, while enhancing anti-tumor cytotoxicity in vitro and in ovarian cancer xenograft experiments. Mechanistically, JNK knockdown led to elevated levels of granzyme B expression which could be recapitulated through overexpression of NFATc1, suggesting an NFATc1 dependent mechanism of action.
Conclusions:
JNK signaling is a significant regulator of CAR-T cell cytotoxicity, offering a potential strategy to directly enhance CAR-T effectiveness in human cancer therapies.
Insights
Targeting c-Jun N-terminal Kinases (JNK) enhances chimeric antigen receptor T (CAR-T) cell therapy against solid tumors. JNK inhibition boosts CAR-T cell anti-tumor activity by modulating nuclear factor of activated T cells (NFAT).
Area of Science:
- Immunology
- Cancer Biology
- Cell Therapy
Background:
- Chimeric antigen receptor T (CAR-T) cell therapy shows promise for solid tumors.
- Nuclear factor of activated T cells (NFAT) is crucial for T cell function.
- Targeting c-Jun N-terminal Kinases (JNK) may enhance CAR-T cell efficacy.
Purpose of the Study:
- To investigate the role of JNK in CAR-T cell function.
- To determine if inhibiting JNK can improve CAR-T cell anti-tumor activity.
- To elucidate the mechanism by which JNK affects CAR-T cell performance.
Main Methods:
- Developed lentiviral short-hairpin RNA (shRNA) for JNK knockdown in CAR-T cells.
- Generated HER2-targeting CAR-T cells from human peripheral blood.
- Assessed functionality in vitro and in human ovarian cancer xenograft models.
Main Results:
- JNK knockdown suppressed antigen-induced stimulation and cytokine production.
- JNK inhibition enhanced in vitro and in vivo anti-tumor cytotoxicity.
- JNK knockdown increased granzyme B expression via an NFATc1-dependent pathway.
Conclusions:
- JNK signaling significantly regulates CAR-T cell cytotoxicity.
- Inhibiting JNK represents a potential strategy to enhance CAR-T cell effectiveness.
- This approach could improve outcomes for patients with human cancers.
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